Assays for identifying agents which affect regulators of UCP3 gene expression
The invention relates to regulators of UCP3 gene transcription, including novel UCP3 transcriptional promoters. UCP3 promoters are used in diagnosis and pharmaceutical development. In particular, transfected cells comprising UCP3 transcriptional regulators operably linked to a reporter are used in high-throughput pharmaceutical screens.
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1. Field of the Invention
The field of this invention is the transcriptional promoter of the UCP3 gene and its use in drug screening.
2. Background
A mitochondrial protein called uncoupling protein (UCP1) is thought to play an important role in the body's regulation of energy utilization. Such regulation provides wide spread physiological controls including body weight, appetite, glucose metabolism, temperature, immune responses, etc. Mechanistically, UCP1 is thought to create a pathway that allows dissipation of the proton electrochemical gradient across the inner mitochondrial membrane in brown adipose tissue, without coupling to any other energy consuming process (for review, see Nicholis & Locke (1984) Physiol Rev 64, 1-64). Unfortunately, the role of UCP1 in physiologies such as body weight regulation in large adult mammals such as people, cattle, pigs, etc. is likely to be limited, since there is little brown adipose tissue in such animals.
UCP2 is a second, related uncoupling protein that is much more widely expressed in large adult mammals (see, e.g. Fleury et al. (1997) Nature Genetics 15, 269-272 and Tartaglia et al. (1996) WO96/05861). Consistent with a role in the regulation of energy utilization generally, and in diabetes and obesity in particular, the UCP2 gene is upregulated in response to fat feeding and maps to regions of the human and mouse genomes linked to hyperinsulinaemia and obesity. Accordingly, upregulators of this gene hold great therapeutic promise for these diseases. To provide regulators of UCP2 gene expression, we cloned the endogenous promoter of the human UCP2 gene and identified various deletion mutants having transcriptional regulatory activity (U.S. Ser. No. 08/846,012, filed Apr. 25, 1997).
UCP3 is a third, related uncoupling protein also widely expressed in large adult mammals. Accordingly, upregulators of this gene hold great therapeutic promise for diseases such as hyperinsulinaemia and obesity. To provide regulators of UCP3 gene expression, we have cloned the endogenous promoter of natural UCP3 genes and identified various deletion mutants having transcriptional regulatory activity.
SUMMARY OF THE INVENTIONThe invention provides methods and compositions relating to the UCP3 gene transcriptional promoter. The compositions include recombinant regulators of gene expression comprising the UCP3 promoter of at least one of SEQ ID NOS:1 and 2, or a deletion mutant thereof at least 50 bp in length having cis transcriptional regulatory activity. Exemplary such deletion mutants comprise at least one of SEQ ID NO:1, bases 411-460, bases 461-510, bases 401-563, bases 319-326, bases 98-104, bases 49-56, bases 49-104 and bases 547-554. In preferred embodiments, the regulators comprise at least one of a GC/SP1, GH-TRE and PR/GR binding site. In further embodiments, the regulators comprise a 5' untranslated UCP3 gene exon. Frequently, the regulators may further comprise a UCP3 or non-UCP3 core promoter operatively joined to said mutant.
The invention also provides hybridization probes and replication/amplification primers having a hitherto novel UCP3 specific sequence contained in SEQ ID NO:1 or 2 (including its complement and analogs and complements thereof having the corresponding sequence, e.g . in RNA) and sufficient to effect specific hybridization thereto (i.e. specifically hybridize with the corresponding SEQ ID NO:1 or 2 in the presence of genomic DNA). Such primers or probes are at least 12, preferably at least 24, more preferably at least 36 bases in length.
The invention also provides cells and vectors comprising the disclosed UCP3 regulators, including cells comprising such regulators operably linked to non-UCP3 genes. Such cells find used in the disclosed methods for identifying agents which regulate the activity of a UCP3 promoter. In an exemplary such method, the cells are contacted with a candidate agent, under conditions wherein, but for the presence of said agent, the gene exhibits a first expression; detecting the presence of a second expression of the gene, wherein a difference between said first and said second expression indicates said agent regulates the activity of a UCP3 gene promoter.
The invention also provides other assays for transcriptional regulators including transcription complex formation assays. An exemplary such assay involves combining a DNA comprising a disclosed regulator with a transcription factor and a candidate agent, under conditions wherein, but for the presence of said agent, the regulator and transcription factor form a first association; detecting the presence of a second association of the regulator and transcription factor, wherein a difference between the first and second associations indicates the agent modulates the association of a UCP3 promoter and transcription factor. The subject nucleic acid regulators also find a variety of other applications, including uses in diagnosis. In particular, hybridization probes and PCR primers derived from the disclosed promoters are used to identify genetic mutations in samples comprising a UCP3 gene.
BRIEF DESCRIPTION OF THE FIGURESFIG. 1. Diagram of mUCP3 promoter constructs driving expression of luciferase enzymatic activity in CaPO.sub.4 transfected HeLa cells. Cells are harvested 18 hrs post transfection and assayed for luciferase.
FIG. 2. Diagram of hUCP3 promoter constructs driving expression of luciferase enzymatic activity in CaPO.sub.4 transfected HeLa cells. Cells are harvested 18 hrs post transfection and assayed for luciferase.
DETAILED DESCRIPTION OF THE INVENTIONThe subject nucleic acids are of synthetic/non-natural sequences and/or are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. Nucleic acids comprising the nucleotide sequence of SEQ ID NO:1 or 2, or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is sometimes advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.
The subject nucleic acids find a wide variety of applications including use as hybridization probes, PCR primers, therapeutic nucleic acids, etc.; use in detecting the presence of UCP3 genes and gene transcripts, in detecting or amplifying nucleic acids encoding additional UCP3 homologs and structural analogs, in gene therapy applications and in a variety of screening assays.
In diagnosis, UCP3-promoter specific hybridization probes find use in identifying wild-type and mutant UCP3 alleles in clinical and laboratory samples. Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. In therapy, therapeutic UCP3 nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active UCP3. For example, UCP3 nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active UCP3 protein. UCP3 inhibitory nucleic acids are typically antisense: single-stranded sequences comprising complements of the disclosed natural UCP3 transcript sequences, particularly the untranslated exon 1. Antisense modulation of the expression of a given UCP3 protein may employ antisense nucleic acids operably linked to gene regulatory sequences. Cells are transfected with a vector comprising a UCP3 sequence with a promoter sequence oriented such that transcription of the gene yields an antisense transcript capable of binding to endogenous UCP3 encoding mRNA. Alternatively, single-stranded antisense nucleic acids that bind to genomic DNA or mRNA encoding UCP3 protein may be administered to the target cell, in or temporarily isolated from a host, at a concentration that results in a substantial reduction in expression of the targeted protein. An enhancement in UCP3 expression is effected by introducing into the targeted cell type UCP3 nucleic acids which increase the functional expression of the corresponding gene products. Such nucleic acids may be UCP3 expression vectors, vectors which upregulate the functional expression of an endogenous allele, or replacement vectors for targeted correction of mutant alleles. Techniques for introducing the nucleic acids into viable cells are known in the art and include retroviral-based transfection, viral coat protein-liposome mediated transfection, etc.
The invention provides efficient methods of identifying pharmacological agents or lead compounds for agents active at the level of UCP3 gene transcription. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. A wide variety of assays for transcriptional regulators are provided including cell-based transcription assays, promoter-protein binding assays, etc. For example, the disclosed luciferase reporter constructs are used to transfect cells such as HeLa cells for cell-based transcription assays. Specifically, HeLa cells are plated onto microtiter plates and used to screen libraries of candidate agents for lead compounds which modulate the transcriptional regulation of the UCP3 gene promoter, as monitored by luciferase expression. An exemplary promoter-protein binding assay is described below. The following examples, exemplary promoter deletion mutants and screening assays are offered by way of illustration and not by way of limitation.
EXAMPLESTransfection of cultured HeLa cells:
Transient transfections were carried out using cultured HeLa cells by calcium phosphate precipitation. 5 ug of promoter-luciferase plasmid DNA were co-transfected with either 1 ug of pMSV expression vector or 1 ug of pMSV-TR expression vector. Samples were co-precipitated with 2 ug of salmon sperm DNA and 0.2 ug of a .beta.-galactosidase internal control expression vector, then applied atop adherent HeLa cells in 6 well tissue culture plates. After 16 hr cells were washed in phosphate buffered saline and refed with fresh DMEM/F12 culture medium supplemented with 10% fetal bovine serum. After an additional 24 hr cells were harvested, lysed and assayed for luciferase and .beta.-galactosidase enzymatic activity according to manufacturer's recommendations (Promega).
Isolation of human and mouse UCP3 genomic clones.
Genomic clones containing the promoter region, the first exon and the remaining 5' untranslated region of the human and mouse UCP3 gene were obtained by hybridization screening of bacteriophage 1 libraries using PCR amplified probes derived from hUCP3 and mUCP3 encoding sequences. The clones were further confirmed by rehybridization using PCR probes derived from 5' untranslated region sequence, which were obtained from RACE PCR amplification. Genomic clones were subcloned into pBluescript KSII (Stratagene), and then sequenced using an Applied Biosystems DNA sequencer. The promoter sequences were subjected to BLAST search on the NCBI server; no homologies to any known sequence were found. Indentically conserved oligonucleotides (see alignment, Table 1) are used in primers and probes for UCP3 genes.
The DNA sequence of the first untranslated exon and upstream DNA of the human and mouse UCP3 genes are shown in SEQ ID NOS:1 and 2, respectively. A number of transcription factor binding sites, splice sites and transcriptional start sites for the human and mouse genes are are shown in Tables I and II, respectively.
TABLE I
______________________________________
Human UCP3 gene transcriptional start,
splice and factor binding sites.
SEQ ID NO: 1,
SITE nucleotides
______________________________________
c-Myc 1132-1138
IBP-1 1355-1360
C/EBP 1006-1013
NF-IL6 266-274
GH-CSE2 843-849
HNF5 566-572
AP-1 1944-1950
START SITE 1461, 1399-1548
HiNF-A 1115-1121
AP-2 961-968
HC3 269-274
GCF 396-403
GH-CSE1 853-859
GR 602-1607
AP-2 1525-1532
INTRON I 1549-2000
______________________________________
TABLE II
______________________________________
Mouse UCP3 gene transcriptional start,
splice and factor binding sites.
SEQ ID NO: 2,
SITE nucleotides
______________________________________
c-Myc 4716-4722
gamma IRE 4851-4859
PR 4861-4869
C/EBP 4287-4295
SRF 3915-3925
NF-IL6 3204-3214
HiNF-A 2968-2976
AP-1 2410-2418
Insulin-Responsive 1152-1159
AP-2 293-301
ApoE-B2 5381-5393
EXON 4935-5080
MyoD 4675-4681
NF-kB 4701-4712
NFIL6 4405-4414
MyoD 3929-3935
AP-2 3706-3714
p53 3062-3072
b-.alpha.-tabuli 2801-2810
GH-CSE1 1974-1982
CREB 791-799
GcF 4996-5003
START 4935-4948
INTRON 5081-5436
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Deletion mutant construction and activity analysis
The promoter activity of the 5' flanking region of human UCP3 gene and a variety of deletion mutants thereof are conveniently screened in a transient transfection assay using mammalian cell lines. An exemplary assay is the HeLa-cell based luciferase reporter assay of FIGS. 1 and 2. Selected promoter deletions are amplified by PCR using targeting primers. Amplification primer pairs for exemplified deletions are as follows:
__________________________________________________________________________
Tagged/untagged
Nucleotide
endonuclease siteSequence
__________________________________________________________________________
att-Mlu1 ATTACGCGT
- att-Hind III ATTAAGCTT
- att-EcoR1 ATTGAATTC
- Mlu1 (core)CGCG
-
H1:
att-Mlu1- (SEQ ID NO:1, nucleotides 1-20)
att-Hind III (reverse compliment of SEQ ID NO:1, nucleotides 1981-2000)
- H2att-Mlu1- (SEQ ID NO:1, nucleotides 1-20)
(reverse compliment of SEQ ID NO:1, nucleotides 1529-1548)
- H3att-Mlu1- (SEQ ID NO:1, nucleotides 200-219)
(reverse compliment of SEQ ID NO:1, nucleotides 1529-1548)
(SEQ ID NO:1, nucleotides 1091-1110)
(reverse compliment of SEQ ID NO:1, nucleotides 1529-1548)
- H5att-Mlu1 (SEQ ID NO:1, nucleotides 1286-1306)
(reverse compliment of SEQ ID NO:1, nucleotides 1529-1548)
(SEQ ID NO:1, nucleotides 1462-1482)
(reverse compliment of SEQ ID NO:1, nucleotides 1529-1548)
(SEQ ID NO:1, nucleotides 1-20)
(reverse compliment of SEQ ID NO:1, nucleotides 1068-1090)
(SEQ ID NO:1, nucleotides 1286-1306)
(reverse compliment of SEQ ID NO:1, nucleotides 1441-1461)
(SEQ ID NO:2, nucleotides 1-25)
att-Hind III - (reverse compliment of SEQ ID NO:2, nucleotides 5411-5436
(SEQ ID NO:2, nucleotides 1-25)
att-Hind III - (reverse compliment of SEQ ID NO:2, nucleotides 5054-5080)
(SEQ ID NO:2, nucleotides 3751-3778)
att-Hind III - (reverse compliment of SEQ ID NO:2, nucleotides 5054-5080)
(SEQ ID NO:2, nucleotides 3940-3967)
att-Hind III - (reverse compliment of SEQ ID NO:2, nucleotides 5054-5080)
(SEQ ID NO:2, nucleotides 4581-4612)
att-Hind III - (reverse compliment of SEQ ID NO:2, nucleotides 5054-5080)
(SEQ ID NO:2, nucleotides 4840-4867)
att-Hind III - (reverse compliment of SEQ ID NO:2, nucleotides 5054-5080)
(SEQ ID NO:2, nucleotides 4930-4958)
att-Hind III - (reverse compliment of SEQ ID NO:2, nucleotides 5054-5080)
__________________________________________________________________________
The deletions may be recombined in any desired variation. For example internal deletions are readily prepared by amplifying both 5' and 3' deletions followed by ligation. Alternatively, a UCP3 promoter deletion may be fused with non-UCP3 promoter element(s) to form heterohybrid promoters. Internal deletions and heterohybrid constructs are exemplified as follows:
M8.multidot.1a&1b pair; 2a&b pair
(SEQ ID NO:2, nucleotides 1-25)
(reverse compliment of SEQ ID NO:2, nucleotides 3727-3751)
(SEQ ID NO:2, nucleotides 4840-4870)
(reverse compliment of SEQ ID NO:2, nucleotides 5054-5080)
(SEQ ID NO:2, nucleotides 3249-3274)
(reverse compliment of SEQ ID NO:2, nucleotides 3727-3751)
(SEQ ID NO:2, nucleotides 4840-4870)
(reverse compliment of SEQ ID NO:2, nucleotides 5054-5080)
- M10 att-Mlu1- (SEQ ID NO:2, nucleotides 1-25)
att-EcoR1- (reverse compliment of SEQ ID NO:2, nucleotides
3727-3751)
(SEQ ID NO:2, nucleotides 3940-3967)
att-EcoR1- (reverse compliment of SEQ ID NO:2, nucleotides
4910-4935)
- M12 1a. att-Mlu1- (SEQ ID NO:2, nucleotides 3940-3967)
(reverse compliment of SEQ ID NO:2, nucleotides 4823-4842)
(SEQ ID NO:2, nucleotides 4863-4887)
(reverse compliment of SEQ ID NO:2, nucleotides 4910-4935)
- M13 &M14 - Oligo anealing no PCR
(SEQ ID NO:2, nucleotides 4843-4862)
(reverse compliment of SEQ ID NO:2, nucleotides 4843-4862)
The PCR fragments are restriction enzyme digested by MluI and HindIII, and then subcloned into MluI and HindIII sites of pGL-2B or pGL-2P (Promega). Transient transfections are carried out using cultured HeLa cells by calcium phosphate precitation method. After 40 hours, cells are harvested, lysed and assayed for luciferase activity. Exemplary mutants are shown to a range of transcriptional activity (FIGS. 1, 2).
Protocol for AP-2 - UCP3 gene promoter binding assay.
A. Reagents:
Neutralite Avidin: 20 .mu.g/ml in PBS.
Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hr, RT.
Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM BME, 1 mg/ml BSA, cocktail of protease inhibitors.
.sup.33 P AP-2 10.times. stock: 10.sup.-6 -10.sup.-8 M "cold" AP-2 supplemented with 200,000-250,000 cpm of labeled AP-2 (Beckman counter). Place in the 4.degree. C. microfridge during screening.
Protease inhibitor cocktail (1000.times.): 10 mg Trypsin Inhibitor (BMB # 109894), 10 mg Aprotinin (BMB # 236624), 25 mg Benzamidine (Sigma # B-6506), 25 mg Leupeptin (BMB # 1017128), 10 mg APMSF (BMB # 917575), and 2 mM NaVo.sub.3 (Sigma # S-6508) in 10 ml of PBS.
Oligonucleotide stock: (specific biotinylated). Biotinylated oligo at 17 pmole/.mu.l, UCP3 gene promoter containing AP-2 site: (BIOTIN)- (SEQ ID NO:1, bases 950-970).
B. Preparation of assay plates:
Coat with 120 .mu.l of stock N-Avidin per well overnight at 4.degree. C.
Wash 2.times. with 200 .mu.l PBS.
Block with 150 .mu.l of blocking buffer.
Wash 2.times. with 200 .mu.l PBS.
C. Assay:
Add 40 .mu.l assay buffer/well.
Add 10 .mu.l compound or extract.
Add 10 .mu.l .sup.33 P-AP-2 (20,000-25,000 cpm/0.1-10 pmoles/well=10.sup.-9 -10.sup.-7 M final concentration).
Shake at 25C for 15 min.
Incubate additional 45 min. at 25C.
Add 40 .mu.l oligo mixture (1.0 pmoles/40 ul in assay buffer with 1 ng of ss-DNA)
Incubate 1 hr at RT.
Stop the reaction by washing 4.times. with 200 .mu.l PBS.
Add 150 .mu.l scintillation cocktail.
Count in Topcount.
D. Controls for all assays (located on each plate):
a. Non-specific binding (no oligo added)
b. Specific soluble oligo at 80% inhibition.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
__________________________________________________________________________
# SEQUENCE LISTING
- - - - (1) GENERAL INFORMATION:
- - (iii) NUMBER OF SEQUENCES: 2
- - - - (2) INFORMATION FOR SEQ ID NO:1:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2000 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: DNA (genomic)
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:1:
- - AAGCTTTGCA CTTGAACATC CATGCTTCTG ACCACCTGCC CTGTGACGCT GG -
#CTCTGTGC 60
- - CCCAGTCCAG AAAAGACTTC TGCCTACTCC TCCTCTGCCC TACCCAGTTA AC -
#TCCCTTTC 120
- - CTTCCCTCCC TTCTGCTTCT CACTCCTCCC CTCCCTTCTC TTCTTCTTCT CC -
#CCTTCCCC 180
- - CATCACCTGG GGCCCGATTC AGCTGTGCCC AGCCCTTACT CTGAGTGCCC AC -
#AGATGGAG 240
- - CCTCCAGTAG CTTCTGTGGG GCACCCTTCC ACCAGGTCCC AGCTCCCTTG GC -
#TCCAGCAG 300
- - TGTCCATGCT AAAGCCTCCA AGTGTCATGT TGGAGAGAAT GGTGTTCACA GT -
#AGATAAGC 360
- - CCAAAATGCC TTACAGTTTA CAGGCTGGAG TCAGGCCCCG CCACGTTGCT GG -
#CTACATGA 420
- - CTTCCCTGAG ATTCCATTTC CTCCTCAGTA AAATAAGTGG TAAGATTTTA GG -
#ATCCCCAG 480
- - CACTAAAAAG AAACGAAATA CTGATACAGG CTCCAACATG GATGAATTTT GA -
#AAGCATTA 540
- - CTATACTAAG TGAAAGAAGC CAGTCACAAA CAAGCACATA TTGGATGATT CC -
#ATTTCTAG 600
- - GAAGTGTTCA GAACAGGCAA ATTTATAGAG ACAGAAAGTA GATTGATTAG TG -
#GTTGCCTG 660
- - AGGCTGGGGA GCGGGGGAAG GGAGGTGACT ACCAATGTGT ATGGAGTTTT TC -
#CAGGGTGA 720
- - GAGGGTGATG AAAATGTTCT AAAATAGATT GTGTTGATGG TTGTGCCACT CA -
#GAATATAC 780
- - TAAAAACCAT TTGAATTGTG CACTTGAAAC AGATGAATTG TACGGTATGT GA -
#ATTCTATA 840
- - TCAATAAATC TGTAATTTAA AAAAAAAAAA TTAGGTCGGG TGCAGTGGCT CA -
#CACCTATA 900
- - ATCCCAGCAC TTTGCCAGAC TGAGGCAGGA GGATCACTTA AGCCCAGGAG TT -
#CAAGACCA 960
- - GCCTGGGGAA CACAGCAAGA CCTCGTCTCT ACTAAAAAAT TTTAAATTAC AA -
#AAAAAAAA 1020
- - AGTAAAAAAA ATAGAATCCT AATAGTACCT ATCTCATAGG ATTGTGGAAA AT -
#AGTAGTAA 1080
- - TGTATGTAAA ATATTTAGCA CATAGTAGGC ACAAAGAAAT GACATTATTA TT -
#AAGAGACC 1140
- - TGGGAGAGCT GTGCCCAGCC TATCGTGGGA GGCCTTGACC TTTGGACTCA AA -
#AGTGGCAG 1200
- - CAGGTCCACC CCCCCATACA CCCTTGTCAC CAAGGAAGCG TCCACAGCTT AA -
#AGGAGCTA 1260
- - TATTAAAGCA CCCCAAGTCA AGAGGACTGA ACCAGATCTG GAACTCACTC AC -
#CTCCCCTC 1320
- - TCACCTCACT GCCCTCACCA GCCAGCCTCT TGTCAAGTGA TCAGGCTGTC AA -
#CCAACTTC 1380
- - TCTAGGATAA GGTTTCAGGT CAGCCCGTGT GTATAAGACC AGTGCCAAGC CA -
#GAAGCAGC 1440
- - AGAGACAACA GTGAATGACA AGGAGGGGCC ATCCAATCCC TGCTGCCACC TC -
#CTGGGATG 1500
- - GAGCCCTAGG GAGCCCCTGT GCTGCCCCTG CCGTGGCAGG ACTCACAGGT AA -
#GACCGGTT 1560
- - TCTCCTCCCT CATCCCTTCC CCTCTCCCTC TCCCTTCTCC TTGTTCTCCC TT -
#TCATTGGA 1620
- - GGCTTTCAGA GAGCAGCCCC GAGCAGTCAG GGCTCACTAG CTGCAGCTTG TC -
#AGACCTGA 1680
- - TAGAGATTCA GTCCAGCCGC CACCTTATGA AAAGGGAGCT GTGGCCTTGA TG -
#AGGGTACT 1740
- - GTGGCAGGGC TGGGGCTTGA ACCCAACACC CGTGTCACTC ACTCAAGACT CA -
#CACCCCCT 1800
- - TTGCCTTGCT GGCTGCCTCT GGTGGGATTT TGCAAATCCC CATAGACAGG AA -
#GTGGCTTT 1860
- - CTTCTTTGCC TGCCCCAGAA TCTCTGCGAT TCCTCCAGAG CATAAATCCC TC -
#TCTTTCCA 1920
- - TGAGGACCCT GGGGCCCTCT TCCTGAGTAG GGATGACAGG GGCACTTCTG AC -
#CTGAGGCG 1980
- - TGGTCCAGGT CATTTGCTGG - # - #
200 - #0
- - - - (2) INFORMATION FOR SEQ ID NO:2:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5436 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: DNA (genomic)
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:2:
- - ACTAGTGACC CAAAAGATCT GTCTATAGCT ACAGCCAGAG CACCGAATGG GA -
#CAAGTATC 60
- - CTCTAAACAC CAAAGATCTC AGGATCTTGA GATGAGGCAC ACAGAAGCTT AA -
#AAGTCTCT 120
- - CAGAGAACCC AAAGGGTATA TCCACAGTAG ACCACTACCA TTGGTTCTAG TG -
#GAATGAAT 180
- - GAGGCATTTA CTGCAGAGGC AGTCTCCTTC CAACCTGATC TAAACCAGAT CT -
#TCATGACC 240
- - CAGTCCTACC CATTCACTGG TCTCATAGCA TCCGAGCCCT CGTCTTGACC TG -
#GCCTGCCC 300
- - GGGGTTGTGT ATTGAAGCTC TAGTTCATGC CCTCAGAAGT GCCTGGTCTG GA -
#GCAGAGGG 360
- - CAATATGGAC ACTGGCCTCT TGGCCCTCCC CTGGCCTGAG GTGTTACTTT AG -
#AAGGAAGA 420
- - ACTGTGAACC AAAAACAACA CCCTTCCTGT TAGCCTAGAC CTCCCCAGAA CA -
#CAGAGCTG 480
- - TACCTCCAGG CTCTGCCAAG CATCCTAGCT TGACACTGCT TATTCTTGGC CA -
#CCAGGAAG 540
- - GCTTGCCTAA GGGCCTGCTC GGCTTGCCCA CCTCCCCCTC AGTGCTTAGA GA -
#TCTGGACT 600
- - GACAGGGATA GTCCTGACTA TTTGCCCAGC CCCCTTGGCC ATGGTTAGGA AA -
#CTAGCACC 660
- - CAAGCAGCAG AGAGGACACA AGCTCATTCC CCTTACCCCA TTCTTTGGGT AG -
#GTCACTGT 720
- - GTCTTGCCTG TTTTACTCTG AGCTGTGAAG CCAGGATCTG CTGTCATTTC TG -
#CCTCCTGT 780
- - TGACGTGTAT TGTTCTATAA AAAGGAATAA TGGGATTTCT AAGTGTGCTC AT -
#CCTTGACC 840
- - CTCTCTAGCC AGGCCTGACC TTTCTTGTTA GCAGGCTCAC GGGGTACAAG CT -
#GGAGAGGT 900
- - GGAACAGTGT ATTAGGGTCC GTCAGCATGG GAACAAAAAA GATCTACCGG CC -
#TGTGGGGA 960
- - CAGCCTCCGT TTCTTTCTGC ACTGGCTCTT CCTGCCTGGC CTCCATCTCT TT -
#CCTAGGGG 1020
- - CCTCATTCTC AGCCCACCAG GCAACTTTGC TATGTAGATC TGAGCCCTTC AG -
#CACAGGAA 1080
- - CCTGACAAGA TACCAAAAAG GGGAAAGTGG CTGAGCACAC GGCTTTGTCT AT -
#GGTAGCTG 1140
- - CTCAAGTGGG ATTTCCACCT GCCTCAGCTC TGCCACTCAG AGCTGCACGA CC -
#TTGAACTA 1200
- - ACTAAGTGCA CCTGACCTTG TGATGTCATG GCCAGGGTTC CACAAGACAG GG -
#TGTATGAG 1260
- - GTATTTGCAA ACCAGGGACC TAGAGAAAAT GTTAGCCCAA GGACCAGACT CG -
#CCTCTGCA 1320
- - ATGCTGTATC CTCAGGACTA ATCTAGATGA CTTCCTTCTC TCCAGGACAA GA -
#GTGAAGCC 1380
- - CACCAGGGGG AGCCCTTGCC CAGAGGCCTT GTCGCCCCAG ACCTTACTCC AT -
#CCCTCCCA 1440
- - ACCTTCCTGA GGAAGAGACT CGCAGGATTG CACGGATATT TTCTTCCCAG TA -
#TTCCAAAA 1500
- - AGACTGAGGA AACCTGAGGG ACGTGGCTAC CAGCCAGCCT CTCAGCTCTG TA -
#AAGCTTGC 1560
- - AAGAGACAAC AGGCCTATTT CCCTCAGCAT CCTCAGGCCA CTAGCATCAC CC -
#TATTCCAT 1620
- - GGGTATCTTG AGACCTAATC AGCTAAGACC AGAGGAGTGT CCCAAACCAC CT -
#AGCAGCTG 1680
- - CCTACCAGGA CCTCAGCTTC CTTCTGTAAT GAGGTGACTG CTGGAAGTGA GA -
#CTCAAGCA 1740
- - CCCAAGTGTC CCACCCATCT TCTGACTGTG GGGCCTAAGG AGGCCTCTCT GA -
#GCAGAGAA 1800
- - ACCAGTGTCC TTGACACAGC CTACTGCTGA CCAGAGCCCA CCTTCTGCTT AA -
#ATAGGACA 1860
- - GGCTATTTTT GCCATAAATA TTGGAAGAAA CATGGAATAA GTATGTACAT AG -
#CTGAGAAA 1920
- - ATTCCAGAGC CCTGTCCAGC TTGTTTGTTT GGGAATGAGG TATTTTATTG AT -
#ATAAATTA 1980
- - TTTTTATGAT GTATTAATCA ATAGAACAGG GGAATTGCCT CCTTTAAAAC TG -
#TATTCGGT 2040
- - TGTCTGAAAT TTAACCATGT TTTTAACATA ATGTTTGTTT CTACTCAGCT CT -
#GAAATTCA 2100
- - TTGCTCTTGC CTTTGTTTTT AATAAAAGTC TGGCATTTGT ATTTGTGTAC AA -
#GTGTTTTT 2160
- - TGGGTTGGTC ACTGGGAAAA GGTGTTGCAG AAAGAAGGAT ACAGCCATCA CT -
#CCACCACC 2220
- - TGCAACATGA GTCGCCATGA GACTTGCTAG TTCTTCCAAC TGTCAGATTG AC -
#CCATGTTA 2280
- - GAGGGATGCT AGTGTTCCAC TTCTGGTGGT TTGGGCTTTT GCTTGCTTGC TT -
#GGTTTGTC 2340
- - TTTTCCTCTG ATGGCCCTTT AAATCTTGCT CAGCACAGTG AGCGTCCAAC TA -
#AGATCTCG 2400
- - TCCCCGGTGT GACTCACGAG GGCACTTCTG TCAGAACACA GCCACTTGGG CA -
#GCTACCAT 2460
- - AGACAAAGTC CTCTGAGGAT AATCTGAACG GAATAGATGT CCAGTGCGTC TG -
#GTAGTGAG 2520
- - GCGGAAAGGA GCTACCTGAG GGCTCTGCTT GTCACCCTGT GTGTCATTTC TC -
#TAAATCCT 2580
- - CACAACTCTA TGATGCCCCT CCCACAATTA CCCTCAACAC CATGGAAGTC GG -
#ATGCATTG 2640
- - CTTCTGTATC GGTACAAAGT GCATCAGGAC CGAAGACATA CCCCAGAGAT AA -
#GGACTCAG 2700
- - ACCAAAGCAG GATACAGGCA GTAAACGCCC TGAATTCGGG CGGTCTCAGG CT -
#GCTAAATC 2760
- - CAGGGAGGGA TTTCTGGAAG AAACCCGTAC TGGGGTAAAG CCCAGAGAAT GG -
#TATAGACC 2820
- - AGGTCATGGA CAGCTGCAAA GAGAACCAGA GAATGTTCTG GACCATATCA GT -
#GTCCAGCC 2880
- - TGGCTGAAAC TGTGGCTCTG GCCTCATCTG GAAGGCACAG ACACGTAGAC TC -
#TGGCTTCG 2940
- - TATGGTCGCT GTGAAGATCA AATGGGACAT TTCTGAAAAT GCTTCGCTGG CA -
#TTTGGCAC 3000
- - ATAATGGAAG CTTGAGAAAT GTCAGCCCTT CCCACTCTTC CTAAGCACCG GG -
#GTGAATTA 3060
- - GAGGCAAGCT CACTGGCTAC CCAGAACGCA GAGCACTCCA GCCTCCCATA AG -
#GGCCATGA 3120
- - ACCTTCAGCC AAACCTGCCC CTCAGATCTC TGTTCCTTGG AGGTCACCTT GC -
#AAACCTGC 3180
- - CCAGTCCTTA GCCAGGGGTT CCCCTTGCCC CAGGCCTTGC CTTCGGCCAG AA -
#CAGAGCTT 3240
- - CCAGTACCTG GAGTTCCACG TCCCTCGGGG TAAGAAACTT CTAGGTTCCA AG -
#CCTAAGGG 3300
- - GTAAAGCCTA GGGTGAAGAA GATCACTGCC AAATCCTGAT CCTGTAAATA AC -
#CTGAAGGA 3360
- - GTCAAGTGAG AACAGGGCCA CAGGAGGGCA CGGGCTGCAG ACAAGGTGAA GG -
#TCTGAGAC 3420
- - ACAGACGACA TGCCCAATTT GTTATTTACC AGTCTCTCCC AGTGATAGCT CT -
#GAGGCAAC 3480
- - TTGACTTGCC AAGTTGTACA ACACATTTGT CACCACAACC AGAATGAACC CT -
#GACTTCCT 3540
- - TATGCATGCC CTCCACAGAT GCCTGGAAAG TATTTCTGGT GACCCAGAGA CT -
#GTAGCAGC 3600
- - CAACCTTAAA ACTCCCAGCA GAGGTTTGAC TCTAGCTAGG CCTGAGCTCT CC -
#TCCCTCTG 3660
- - GGTTTCTCCA GAACCTGCTG CCTCTAGTTT GACTTCTACT TGTAGCCCCA GC -
#CCCCACAT 3720
- - GGTTCCCACA GTCCTGCCCA TGTCCAGATT AATAGTTCCA GACTGAGCTC AA -
#AGCAGTGA 3780
- - GCAGGTCCTG GTCCAAGAAA CAGGATAGAC AGTCTAGATG TCACAGCACC CA -
#CCCTGCCA 3840
- - TGGCGCTGCG CTCAAGTTCC AAAATGTCCT CTACCTTCTC TGTCCTACTT AT -
#CTCCTCTC 3900
- - CCCTCTCCTT TTAGTTTCCC TTCTATGCCA CCTGGCTCCC AACTTCAGCT GT -
#GCCCACCC 3960
- - AGTCTTTAAC CTCAGCGCCT GCAGATGGAC CAGCCTACAG TGGTTGCTGC AA -
#GGGATTTT 4020
- - GCTCACCCTG CCCCATCTCC GGTCCCAGCA TGTCCATGTC ATGAGGAATC AG -
#GATTGCAG 4080
- - CTGATAGACC CAAAGTGTGG TATAGGTTAC AGGCAAGTCA GGCCTGCAGT GA -
#CCTAGCTG 4140
- - TCTGTCCTTG ACAGTCAGTT TTCTGTGTAA AGTAAGCATT GACACATGAG GG -
#CTCTACAC 4200
- - AGTACGTCAG TGTCTACCGG TACAGTGCAG TGTGTTAACA CCACTGTGAA AG -
#AAACCAGC 4260
- - CACAAGAGGC TATACATTCT ATATAATCCT ACTCAAACAA AGCATCTAGA AC -
#AAGCAAAT 4320
- - TAAGAAGCAG AATAAAGACG AGTGGTTGCC TGAAGCTGGG GGAAGGGAAA GG -
#GGGTGACT 4380
- - TGTTCTGTTT TTGTCTTTGA GCCATGATGA AAGTGTTCTG AACTAGCATG TA -
#GTAGTTGA 4440
- - TCAACCAGAC TGTACTAAAC ACTATTGTGT GCTCTATGTG GGCAAATCAC AT -
#GCTGTGTA 4500
- - CAAAACATCT GTTGTCCTTT GTTTTTAAAT TTAGGATCCT GCTTCCTAGA GA -
#TGTGGGAA 4560
- - ATAGAAGCGC TGTGCCTGAA ATATCAAGCA TATCTTGGCA CCAAGATGTC CT -
#CTACCTTC 4620
- - TCTGCCCCGT CTTCTCCTCT CCCCTCTTGA GAATGTCAGG CCTCTAAGAA GT -
#GACACCTG 4680
- - TAACCATTGT ATAGGATCCT GGAGAGCCCC TGTCCTAAGA GACCTTGTCC TT -
#TGGGCTCT 4740
- - CAAAGGTGAC AAATGCTGTC ACACACCTCC TGGCCACCAA GGTAGCTCTC CT -
#CTTGAAAG 4800
- - CTCAAAGGAG CCACATTAAA GAGCCCCAGG TCACGGAAGC TAAACCAGAT CT -
#GGAACTCA 4860
- - CTGGTCCCCT CCCCGCAGCC TGCCTCTTGT CAAGTGATCA GACTGTCAAC TA -
#GCTTCTCA 4920
- - GAATTAGGTT TCAGGTCAGC TGGTGCACAG GGCCAGTGCC GAGCCAGGGA CA -
#GCAGAGAC 4980
- - AACAGTGAAT GGTGAGGCCC GGCCGTCAGA TCCTGCTGCT ACCTAATGGA GT -
#GGAGCCTT 5040
- - AGGGTGGCCC TGCACTACCC AACCTTGGCT AGACGCACAG GTAAGACCCC AT -
#ACTCTGCT 5100
- - CTCCTCTCCC TTTTTCCCTT CCATGGATGC TCACAGCCAG GAGCTTGCTG GG -
#ATCACTCA 5160
- - GCACTGCGTG AGAGACCGAG AGTGAGCCGG TCTAGCTCCC ACCTAGTAAA GA -
#TGAAGGAA 5220
- - CTGCAGGCCT GGGGAGGGCC TTGACTTCCA CATCTATGTG ACTCCTCACA AC -
#TCCCGTGT 5280
- - TTTGCTGACT CCTCTGCTGG GATCTTACAA ATGCCAAATG AAAAGTGTCC CT -
#CTCCTTTG 5340
- - GCCCAGGATC CCCACGGAGC ACAAAGCCCT CTCCAGCAAG GATCCTGGGG CC -
#CTTCCTGG 5400
- - GTAAAAATAA TGAGGGCACT CTGGCCTGAA GCCTGG - #
- # 5436
__________________________________________________________________________
Claims
1. A method for identifying agents which modulate the association of a UCP3 transcriptional regulator and a transcription factor, said method comprising steps:
- combining said UCP3 transcriptional regulator, said transcription factor and a candidate agent, under conditions wherein, but for the presence of said agent, said regulator and said transcription factor form a first association, said regulator comprising a recombinant nucleic acid comprising SEQ ID NO:1 or 2, or a fragment thereof at least 50 bp in length comprising at least one nucleotide sequence selected from the group consisting of: SEQ ID NO:1, nucleotides 411-460; SEQ ID NO:1, nucleotides 461-510; SEQ ID NO:1, nucleotides 401-563; SEQ ID NO:1, nucleotides 319-326; SEQ ID NO:1, nucleotides 98-104; SEQ ID NO:1, nucleotides 49-56; SEQ ID NO:1, nucleotides 49-104; SEQ ID NO:1, nucleotides 547-554; SEQ ID NO:1, nucleotides 1-1548; SEQ ID NO:1, nucleotides 200-1548; SEQ ID NO:1, nucleotides 1090-1548; SEQ ID NO:1, nucleotides 1285-1548; SEQ ID NO:1, nucleotides 1-1090; SEQ ID NO:1, nucleotides 1285-1461; SEQ ID NO:2, nucleotides 1-5080; SEQ ID NO:2, nucleotides 3751-5080; SEQ ID NO:2, nucleotides 3940-5080; SEQ ID NO:2, nucleotides 4580-5080; SEQ ID NO:2, nucleotides 4840-5080; SEQ ID NO:2, nucleotides 1-3571; SEQ ID NO:2, nucleotides 3940-4935; and SEQ ID NO:2, nucleotides 4843-4862 and having cis transcriptional regulatory activity; and
- detecting the presence of a second association of said regulator and said transcription factor,
- wherein a difference between said first and said second association indicates that said candidate agent is an agent which modulates the association of said UCP3 transcriptional regulator and said transcription factor.
2. The method of claim 1, wherein the nucleic acid comprises at least one nucleotide sequence selected from the group consisting of: SEQ ID NO:1, nucleotides 411-460; SEQ ID NO:1, nucleotides 461-510; SEQ ID NO:1, nucleotides 401-563; SEQ ID NO:1, nucleotides 49-104; SEQ ID NO:1, nucleotides 1-1548; SEQ ID NO:1, nucleotides 200-1548; SEQ ID NO:1, nucleotides 1090-1548; SEQ ID: NO:1, nucleotides 1285-1548; SEQ ID NO:1, nucleotides 1-1090; SEQ ID NO:1, nucleotides 1285-1461; SEQ ID NO:2, nucleotides 1-5080; SEQ ID NO:2, nucleotides 3751-5080; SEQ ID NO:2, nucleotides 3940-5080; SEQ ID NO:2, nucleotides 4580-5080; SEQ ID NO:2, nucleotides 4840-5080; SEQ ID NO:2, nucleotides 1-3571; and SEQ ID NO:2, nucleotides 3940-4935.
3. The method of claim 1, wherein the nucleic acid comprises SEQ ID NO:1 or 2, or a fragment thereof at least 50 bp in length comprising at least one nucleotide sequence selected from the group consisting of: SEQ ID NO:1, nucleotides 401-563; SEQ ID NO:1, nucleotides 1-1548; SEQ ID NO:1, nucleotides 200-1548; SEQ ID NO:1, nucleotides 1-1090; SEQ ID NO:2, nucleotides 1-5080; SEQ ID NO:2, nucleotides 3751-5080; SEQ ID NO:2, nucleotides 3940-5080; SEQ ID NO:2, nucleotides 4580-5080; SEQ ID NO:2, nucleotides 4840-5080; SEQ ID NO:2, nucleotides 1-3571; and SEQ ID NO:2, nucleotides 3940-4935.
4. The method of claim 1, wherein the nucleic acid comprises SEQ ID NO:1 or 2.
5. The method of claim 1, wherein the nucleic acid comprises at least one binding site from Table I or II.
6. The method of claim 1, wherein the nucleic acid is operatively joined to a non-UCP3 core promoter.
7. The method of claim 1, wherein the nucleic acid comprises a 5' untranslated UCP3 gene exon.
8. A method for identifying agents which regulate the activity of a UCP3 transcriptional regulator, said method comprising steps:
- contacting a cell with a candidate agent, said cell comprising said UCP3 transcriptional regulator comprising a recombinant nucleic acid comprising SEQ ID NO:1 or 2, or a fragment thereof at least 50 bp in length comprising at least one nucleotide sequence selected from the group consisting of: SEQ ID NO:1, nucleotides 411-460; SEQ ID NO:1, nucleotides 461-510; SEQ ID NO:1, nucleotides 401-563; SEQ ID NO:1, nucleotides 319-326; SEQ ID NO:1, nucleotides 98-104; SEQ ID NO:1, nucleotides 49-56; SEQ ID NO:1, nucleotides 49-104; SEQ ID NO:1, nucleotides 547-554; SEQ ID NO:1, nucleotides 1-1548; SEQ ID NO:1, nucleotides 200-1548; SEQ ID NO:1, nucleotides 1090-1548; SEQ ID NO:1, nucleotides 1285-1548; SEQ ID NO:1, nucleotides 1-1090; SEQ ID NO:1, nucleotides 1285-1461; SEQ ID NO:2, nucleotides 1-5080; SEQ ID NO:2, nucleotides 3751-5080; SEQ ID NO:2, nucleotides 3940-5080; SEQ ID NO:2, nucleotides 4580-5080; SEQ ID NO:2, nucleotides 4840-5080; SEQ ID NO:2, nucleotides 1-3571; SEQ ID NO:2, nucleotides 3940-4935; and SEQ ID NO:2, nucleotides 4843-4862 and having cis transcriptional regulatory activity, said regulator operably linked to a non-UCP3 gene, under conditions wherein, but for the presence of said agent, said gene exhibits a first expression;
- detecting the presence of a second expression of said gene;
- wherein a difference between said first and said second expression indicates that said candidate agent is an agent which regulates the activity of said UCP3 gene transcriptional regulator.
9. A method according to claim 8, wherein said gene is a reporter and said detecting step comprises detecting a colorimetric or luminescent signal of said reporter.
10. A method according to claim 8, wherein said gene is detected by hybridization to a nucleic acid specific for said gene.
11. The method of claim 8, wherein the nucleic acid comprises at least one nucleotide sequence selected from the group consisting of: SEQ ID NO:1, nucleotides 411-460; SEQ ID NO:1, nucleotides 461-510; SEQ ID NO:1, nucleotides 401-563; SEQ ID NO:1, nucleotides 49-104; SEQ ID NO:1, nucleotides 1-1548; SEQ ID NO:1, nucleotides 200-1548; SEQ ID NO:1, nucleotides 1090-1548; SEQ ID NO:1, nucleotides 1285-1548; SEQ ID NO:1, nucleotides 1-1090; SEQ ID NO:1, nucleotides 1285-1461; SEQ ID NO:2, nucleotides 1-5080; SEQ ID NO:2, nucleotides 3751-5080; SEQ ID NO:2, nucleotides 3940-5080; SEQ ID NO:2, nucleotides 4580-5080; SEQ ID NO:2, nucleotides 4840-5080; SEQ ID NO:2, nucleotides 1-3571; and SEQ ID NO:2, nucleotides 3940-4935.
12. The method of claim 8, wherein the nucleic acid comprises SEQ ID NO:1 or 2, or a fragment thereof at least 50 bp in length comprising at least one nucleotide sequence selected from the group consisting of: SEQ ID NO:1, nucleotides 401-563; SEQ ID NO:1, nucleotides 1-1548; SEQ ID NO:1, nucleotides 200-1548; SEQ ID NO:1, nucleotides 1-1090; SEQ ID NO:2, nucleotides 1-5080; SEQ ID NO:2, nucleotides 3751-5080; SEQ ID NO:2, nucleotides 3940-5080; SEQ ID NO:2, nucleotides 458-5080; SEQ ID NO:2, nucleotides 4840-5080; SEQ ID NO:2, nucleotides 1-3571; and SEQ ID NO:2, nucleotides 3940-4935.
13. The method of claim 8, wherein the nucleic acid comprises SEQ ID NO:1 or 2.
14. The method of claim 8, wherein the nucleic acid comprises at least one binding site from Table I or II.
15. The method of claim 8, wherein the nucleic acid is operatively joined to a non-UCP3 core promoter.
16. The method of claim 8, wherein the nucleic acid comprises a 5' untranslated UCP3 gene exon.
- Boss et al, FEBS Letters, vol. 408, No. 1: pp. 39-42, May 12, 1997.
Type: Grant
Filed: Oct 9, 1998
Date of Patent: Nov 2, 1999
Assignee: Tularik Inc. (South San Francisco, CA)
Inventors: M. Catherine Amaral (S. San Francisco, CA), Ning Zhang (S. San Francisco, CA), Jin-Long Chen (S. San Francisco, CA)
Primary Examiner: Terry McKelvey
Attorney: Richard Aron Osman
Application Number: 9/169,203
International Classification: C12Q 168; C12Q 102;
